The spread of the invasive mosquito Aedes albopictus (Diptera: Culicidae) in Portugal: a first genetic analysis

Background Aedes albopictus, commonly known as the Asian tiger mosquito, has become one of the most invasive mosquito species. Over the last 5 decades, it has been introduced and established in various tropical and temperate regions worldwide. First reported in Europe in 1979 in Albania and later in Italy in 1990, the species is now established in 13 European Union (EU)/European Economic Area (EEA) countries and 337 regions (2023). In Portugal, Ae. albopictus was first detected in the Algarve and Penafiel regions in 2017, followed by Alentejo in 2022 and Lisbon in 2023. This mosquito species poses a significant public health risk as a vector for numerous pathogenic viruses, including dengue, Zika, and chikungunya. Methods Aedes albopictus collected in Lisbon in 2023 were analyzed using cytochrome c oxidase I (COX) gene sequencing to understand their genetic relationships. Results Our data indicate that the Ae. albopictus mosquito populations detected in three locations in Lisbon in 2023 correspond to recent but distinct introduction events. Conclusions Although there has been no local transmission of Aedes-transmitted viruses in mainland Portugal to date, the spread of the mosquito and increased international travel increase the risk of Aedes-borne disease outbreaks. The ongoing spread of Ae. albopictus in the country and the confirmed multiple introductions in new locations raise awareness of the need to monitor mosquito vectors to control and prevent autochthonous Aedes-borne disease outbreaks. Graphical Abstract


Background
First described by Skuse in India in 1894, Aedes (Stegomya) albopictus has been recognized as one of the most invasive mosquito species, successfully colonizing numerous tropical and temperate regions worldwide over the last 5 decades.Aedes albopictus was first reported in Europe in 1979 in Albania [1], followed by Italy in 1990 [2].Italy is currently considered the most infested country in Europe, with Ae. albopictus established over large areas and thriving particularly in urban areas [3].Since its introduction in Italy, Ae. albopictus has steadily spread throughout Europe, particularly to most Mediterranean countries.In 2023, Ae. albopictus was established in 13 European Union (EU)/European Economic Area (EEA) countries and 337 regions, while in 2013, it was established only in 8 countries and 114 European regions [4].In Portugal, Ae. albopictus was first detected in 2017 through two different introduction events in the Algarve, the southernmost region [5], and Penafiel, in the Porto region [6].In 2022, this vector was detected in the Alentejo region and, in late, September 2023 in Lisbon [7].The National Vector Surveillance Network-Rede de Vigilância de Vectores (REVIVE) has been running since 2008 under the auspices of the Portuguese Ministry of Health [8].REVIVE conducts nationwide surveillance of the most critical hematophagous arthropods for public health (mosquitoes, ticks, and sandflies).Regular surveillance of mosquito species and screening field-collected mosquitoes for arboviruses is conducted.Airports, ports, storage areas, and certain border regions with Spain are monitored throughout the year with the involvement of local and regional authorities.
In addition to the nuisance associated with establishing Aedes albopictus, its ability to act as a vector for a wide range of arboviruses remains a primary concern.This mosquito species has emerged as a significant global public health threat due to its ability to transmit several pathogenic flaviviruses (such as dengue, Zika, and yellow fever) and alphaviruses (especially chikungunya virus).The central tourist regions in the country are the Algarve, Lisbon, and Porto, where the presence of this vector species can be a significant concern due to the higher risk of incoming viremic travelers.Since 2007, cases of autochthonous transmission of chikungunya associated with Ae. albopictus have been documented in Europe [9].Dengue has been reported in Europe since 2010, with autochthonous cases transmitted by Ae. albopictus in Croatia, France, Italy, and Spain [10].
To date, local transmission of Aedes-borne viruses has not yet been detected in mainland Portugal.However, the geographic expansion of Ae. albopictus across Portugal, combined with the increasing number of international travelers, often from regions with ongoing Aedes-borne outbreaks, highlights the importance of mosquito vector surveillance and control and raises public health concerns about the risk of increased introduction and autochthonous transmission of Aedes-borne viral infections.Here, we report a preliminary genetic analysis of the Ae.albopictus mosquitoes detected in Lisbon in 2023, using the primary barcode sequence for members of the animal kingdom, a partial sequence of the cytochrome c oxidase I (COX) gene, widely used to study the genetic relationships of Ae. albopictus [11][12][13][14] and previously used in Portuguese mosquito populations [15].

Mosquito samples and DNA extraction
Larvae samples were collected in natural and artificial water containers at three sites in the Lisbon region (Algés, Alvalade, and São Domingos de Benfica; Table 1).Mosquito samples from the Algarve and Porto populations, collected in 2022 and 2023, were also submitted for analysis.All mosquito samples were collected by the national REVIVE surveillance network [6] in public and private properties, with the knowledge and permission of the respective responsible/owners.The collected mosquitoes were reared to adults in the insectary (by collection site and date), and 10 adult mosquitoes were randomly selected from each collection site for analysis.Sampled mosquitoes collected in the Lisbon region were ground individually by grinding with a mortar and pestle with liquid nitrogen and further ground after adding 500 µL lysis buffer (NUCLIS-ENS ® easyMAG, Biomérieux).Nucleic acid extraction was performed with the prepared lysate suspensions in the automated platform NUCLISENS ® easyMAG (Biomérieux), as previously described [6].Larval and adult mosquitoes were morphologically confirmed as Ae.albopictus [16,17].

Molecular analysis
Molecular identification was performed using the COX gene of mitochondrial DNA with primers LCO1490 and HCO 2198 [18], as previously described [6].Aedes albopictus haplotype diversity for COX sequences was estimated using DnaSP v.6.10.01 [19] using default parameters.
To integrate the mosquitoes circulating in Portugal and into the global Ae.albopictus genetic diversity, the consensus nucleotide partial sequences of the COX gene were aligned against several sequences available in GenBank (20 COX previously reported at the global level; Fig. 1) using BioEdit version 7.2.5 [20] and further used to construct a phylogenetic tree using the unweighted pair group method with arithmetic mean (UPGMA; 1000 bootstraps) in MEGA X [21].For better visualization of the tree, the KX383935 sequence was used as an outgroup.Figure 1 was generated using BioRender.com.

Results
The 10 partial COX sequences obtained for Ae.albopictus mosquitoes collected at each of the three sites in Lisbon (30 sequences in total) show low genetic diversity, with only one haplotype in Algés (GenBank accession number PP825984) and São Domingos de Benfica (GenBank accession number PP825985).In Alvalade, two haplotypes were identified, with one corresponding to nine collected mosquito specimens (GenBank accession number PP825983) and the other corresponding to only one (GenBank accession number PP825982).Sequences from mosquitoes collected in 2022 and 2023 in the Algarve and Porto regions are identical to previously detected haplotypes for these regions (Table 1; Fig. 1), which were identified as haplotype 1 (the Algarve and Porto) and haplotype 2 for the Algarve, as previously defined [15].The haplotype diversity analysis for the sequences detected in the Lisbon region shows, in Alvalade, two haplotypes, haplotype 2 and haplotype 3, previously detected in the Algarve, and two new haplotypes for the other sites, haplotype 6 in Algés (with similarity to sequences from widespread sites) and haplotype 7 in São Domingos de Benfica, identical to sequences detected in Spain (Table 1 and Fig. 1).

Discussion
The observed low diversity of COX sequences in mosquitoes collected in Lisbon is consistent with recent introductions.Although the collection of immature mosquitoes may bias the population diversity, collections were made in heavily infested containers, corresponding to multiple female mosquito ovipositions.For most of these sites, the water containers containing immature mosquitoes were the only ones found with Ae. albopictus in the surveyed area.Some sites were analyzed following citizen science reports of mosquito presence, and others resulted from detailed inspections by the Lisbon and Tagus Valley Health Authority following the initial detection event.
The COX sequences from the three Lisbon sites differed, so these introductions can be considered separate events.Although preliminary, these data suggest that, at least in Alvalade, the introduction may have come from the Algarve.However, direct external introductions cannot be excluded, especially in São Domingos de Benfica and Algés.Nevertheless, more data are needed on the current mosquito populations circulating in Porto, the Algarve, and Alentejo.A finer genetic analysis, namely by mitogenome sequencing, with a broader sampling of mosquitoes in the coming season (2024) is underway to obtain more details.Nevertheless, the identification of three independent introduction events in one mosquito season highlights the potential of this species to invade new geographic areas in a short period.It also means that these events are likely underway and indicate naive regions that could be rapidly colonized.

Conclusion
In 2022, over 19 million travelers entered Europe from dengue-affected areas [22].Between 2012 and 2022, our team at the National Reference Laboratory of the Portuguese National Institute of Health (INSA) detected 142 Aedes-borne infections in viremic travelers.However, the high rates of asymptomatic infection in humans and the relatively short viremic window of symptomatic patients suggest that many traveler infections may be under-recognized, and the number of viremic travelers is much higher.Europe is experiencing a warming trend, with more frequent and severe heat waves and floods and longer and warmer summers [10].This creates more favorable conditions for invasive mosquito species such as Ae.albopictus and Ae.aegypti.The geographical spread of invasive mosquito species to previously unaffected areas in the EU/EEA is an ongoing reality.In most European countries, cold winters do not allow year-round transmission [10], but in the southernmost region of Portugal, the Algarve, adult Ae. albopictus mosquitoes are already present year-round, although at lower population levels in winter.Given climate change, with continued increases in temperature and subsequent milder winters, the conditions for virus transmission will undoubtedly increase.
Given the ongoing spread of Ae. albopictus in mainland Portugal, it is essential to raise awareness of mosquitoborne diseases among the general public, healthcare professionals, and travelers.

Table 1
Aedes albopictus samples collected in Portugal and included in this study a Number of individually analyzed mosquitoes from the same collection site and date with identical COX sequence UPGMA phylogenetic tree constructed from 18 (9 novel) COX sequences obtained from mosquitoes circulating in Portugal (Table 1) and 20 sequences available in GenBank.Bootstrap values (1000 replicates) greater than 65 are shown above the branches, and branch lengths (in the same units as those of the evolutionary distances used to infer the phylogenetic tree) are shown below the branches.Evolutionary distances were calculated using maximum composite likelihood.Sequences are identified by GenBank accession numbers, country region, country, and year of collection (if available).Tree nodes are identified by the COX haplotype.Colored circles indicate the regions where mosquitoes from Portugal were collected.B Map showing the locations of mosquito sample collection and the most likely origin of introduction in Lisbon, based on available data.Composite figure created with https:// www.biore nder.com/ (See figure on next page.) Fig. 1 (See legend on previous page.)